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CLINICAL CASE SEMINAR |
London Centre for Paediatric Endocrinology and Metabolism (K.H., V.V.S., M.D.), Great Ormond Street Hospital for Children NHS Trust, London WC1N 3JH, United Kingdom, and The Institute of Child Health, London WC1N 1EH, United Kingdom; Faculty of Life Sciences (K.E.C., R.M.S., M.J.D.), University of Manchester, Manchester M13 9PT, United Kingdom; West Midlands Regional Genetics Service (A.L., E.A.M.), Birmingham Women’s Health Care Trust, Birmingham, B15 2TG, United Kingdom, and Section of Medical and Molecular Genetics, University of Birmingham, Birmingham B15 2TT, United Kingdom; Endocrinology and Metabolism Service (S.K., B.G.), Hadassah-Hebrew University Medical Center, Jerusalem, 91120, Israel; and Department of Child Health (J.W.G.), University of Wales, Cardiff CF14 4XN, United Kingdom; Department of Biomedical Sciences (A.S.), Leeds University, Leeds LS2 9JT, United Kingdom; Department of Pediatrics/Genetics (H.T.C., B.B.J., K.B.), Odense University Hospital, 5000 Odense, Denmark; and Section of Medical and Molecular Genetics (E.A.M.), University of Birmingham, Birmingham B15 2TT, United Kingdom
Address all correspondence and requests for reprints to: Dr. K. Hussain, Unit of Biochemistry, Endocrinology, and Metabolism, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, United Kingdom. E-mail: k.hussain{at}ich.ucl.ac.uk.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Patients and Methods: Using patch-clamp techniques on pancreatic tissue obtained at the time of surgery, we investigated the electrophysiological properties of ATP-sensitive K+ (KATP) channels in pancreatic ß-cells in a patient with BWS and severe medically-unresponsive hyperinsulinemic hypoglycemia.
Results: Persistent hyperinsulinism was found to be caused by abnormalities in KATP channels of the pancreatic ß-cell. Immunofluorescence studies using a SUR1 antibody revealed perinuclear pattern of staining in the BWS cells, suggesting a trafficking defect of the SUR1 protein. No mutations were found in the genes ABCC8 and KCNJ11 encoding for the two subunits, SUR1 and KIR6.2, respectively, of the KATP channel. Genetic analysis of this patients BWS showed evidence of mosaic paternal isodisomy.
Conclusions: In this novel case of BWS with mosaic paternal uniparental disomy for 11p15, persistent hyperinsulinism was due to abnormalities in KATP channels of the pancreatic ß-cell. The mechanism/s by which mosaic paternal uniparental disomy for 11p15 causes a trafficking defect in the SUR1 protein of the KATP channel remains to be elucidated.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Histologically, HI can be divided into two major subtypes (9). The diffuse form of the disease is inherited recessively and involves all ß-cells within the pancreas. The focal form (Fo-HI) consists of adenomatous hyperplasia within a limited region of the pancreas, and it is caused by somatic loss of heterozygosity, including maternal Ch11p15 in a ß-cell precursor carrying a germ-line mutation in the paternal allele of SUR1 or Kir6.2 (10, 11). Several imprinted genes are located within this chromosomal region, some of which, including p57Kip2 and IGF-II, have been associated with the regulation of cell proliferation. p57Kip2 is paternally imprinted (i.e. expressed from the maternal allele) in human pancreatic ß-cells, and the loss of expression in Fo-HI is caused by loss of heterozygosity, leading to increased cell proliferation and increased IGF-II expression (12).
Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome that is clinically and genetically heterogeneous. Phenotypically, BWS is associated with prenatal and/or postnatal overgrowth, macroglossia, anterior abdominal wall defects, organomegaly, hemihypertrophy, ear lobe creases and helical pits, and renal tract abnormalities. Genetically, BWS is a complex multigenic disorder caused by dysregulation of imprinted growth regulatory genes within the Ch.11p15 region (13). Approximately 2% of BWS cases have chromosomal abnormalities involving Ch.11p15.5, and 5% of sporadic cases have germ-line mutations in the candidate tumor suppressor gene CDKN1C. About 20% of patients with BWS have paternal uniparental disomy (UPD) for Ch.11p15 (13), and these patients are predicted to have increased expression of the paternally expressed growth promoter IGF2 and reduced expression of the maternally expressed CDKN1C and H19 genes. In all cases with UPD for Ch.11p15, the affected patient is mosaic for a paternal isodisomy and a normal cell line, indicating that paternal UPD has arisen due to a postzygotic event (14) (see Fig. 1
). Up to 60% of sporadic patients have methylation alterations at imprinting control regions, leading to disordered imprinting of IGF2, CNDKN1C, or H19 (15).
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Using patch-clamp techniques on pancreatic tissue obtained at the time of surgery, we investigated the electrophysiological properties of KATP channels in pancreatic ß-cells in a patient with BWS and severe medically unresponsive hyperinsulinemic hypoglycemia. We report a novel case of BWS with mosaic paternal UPD for Ch.11p15 in which persistent hyperinsulinism was found to be caused by loss of function of KATP channels of the pancreatic ß-cell.
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Patients and Methods |
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The patient was born at term with a birth weight of 3.8 kg to nonconsanguineous parents. He developed persistent hyperinsulinemic hypoglycemia within 24 h after birth, with a maximum glucose infusion rate of 20 mg/kg·min (normal is 4–6 mg/kg·min). At birth, there were no obvious clinical features of BWS, but, postnatally, he developed right-sided hemihypertrophy, macroglossia, ear lobe creases, and an umbilical hernia. He failed to respond to diazoxide (5–20 mg/kg·d dose) or nifedipine (0.25 mg/kg·d dose) and had breakthrough hypoglycemia while on continuous sc infusions of octreotide (5–25 µg/kg·d dose) and glucagon (5–20 µg/kg·h dose). Normoglycemia could only be maintained on a combination of continuous infusion of 20% glucose and feeds.
Given the severity of his hyperinsulinemic hypoglycemia, it was decided to perform a partial pancreatectomy. He continued to be hypoglycemic after the operation and again failed to respond to maximum doses of diazoxide but eventually maintained normoglycemia on octreotide injections (20 µg/kg·d) and frequent (two to four hourly) feeds supplemented with 10% Maxijul. Thereafter, it proved possible to progressively reduce the energy content of his diet and to wean down his dose of octreotide such that all therapy was discontinued at the age of 14 months. At that age, a prolonged fast resulted in hypoglycemia (blood glucose, 2.4 mmol/liter at 15 h fasting) associated with an undetectable circulating serum insulin concentration (<1 mU/liter) and a free fatty acid concentration of 2.7 mmol/liter with a total ketone body response of 2.1 mmol/liter. Over the following year, no spontaneous episodes of hypoglycemia have been documented, and he demonstrates normal growth and neurodevelopmental progress. The study was approved by the Ethics Committee of Great Ormond Street Children’s Hospital and the Institute of Child Health; written informed consent was obtained from the parents or guardians.
Molecular genetic analysis for BWS
Evidence of paternal isodisomy was sought by genotyping the patient and his parents with the polymorphic microsatellite markers D11S1984 and tyrosine hydroxylase (TH) in chromosome 11p15.5. Each marker was amplified separately using fluorescently tagged primers described previously (GenBank accession no. G08894) (20, 21). PCR reactions for D11S1984 contained 10 pmol of each primer, 0.2 mM dNTP, 100 ng DNA, 1x PCR buffer (AmpliTaq; Applied Biosystems, Foster City, CA), 1.5 mM magnesium chloride (AmpliTaq), and 0.75 U of Taq DNA polymerase (AmpliTaq) in 10 µl. The PCR cycling was performed using a Tetrad DNA engine as follows: initial denaturation at 95 C for 5 min, followed by 24 cycles of 95 C for 1 min, 57 C for 1 min, 72 C for 1 min, and a final primer extension at 72 C for 5 min. The TH marker was amplified with the cycling parameters described above but with an annealing temperature of 62 C. PCR products were resolved on a 6% denaturing acrylamide gel using the ABI377 (Applied Biosystems) automated sequencer and analyzed using GeneScan Analysis Software (Applied Biosystems). For each marker, the parental origin of each allele was determined by comparison to the parental alleles, and each trace was examined for evidence of a ratio in favor of the paternally derived allele. The peak area obtained was used to calculate a dosage ratio of paternal to maternal allele. A ratio greater than 1.3 in favor of the paternally derived allele is considered to be evidence of mosaic paternal isodisomy.
Histology of resected pancreatic tissue
The pancreas was fixed in 10% phosphate-buffered formalin for 24 h, and blocks were processed into paraffin wax. Sections (4 µm thick) were cut and stained with hematoxylin and eosin. Immunostaining was performed using polyclonal antibodies against glucagon (1:200 in 20% normal goat serum; Dako, Glostrup, Denmark), insulin (1:150 in 20% normal swine serum; Dako), pancreatic polypeptide (1:600 in 20% normal swine serum; Dako), and somatostatin (1:200 in 20% normal swine serum; Dako), as well as using monoclonal antibodies against proinsulin (1:1000 in 20% normal rabbit serum; Novocastra, New Castle, UK) and low-molecular-weight cytokeratin clone MNF-116 (1:100 in PBS; Dako). Visualization was obtained using extravidin biotin peroxidase kit (Sigma, Poole, UK). Antigen retrieval was achieved for somatostatin and MNF-116 by previous digestion with 0.02% protease for 5 min at 37 C and for proinsulin by pressure cooking within a microwave oven at full power under pressure for 4 min in preheated citrate/EDTA buffer (pH 6.2).
Immunostaining for p57Kip2
Antigen retrieval was accomplished by boiling the sections for 15 min in a microwave oven. Slides were blocked by nonimmune serum for 10 min at room temperature before application of each primary antibody. Slides were double stained for p57Kip2 (Santa Cruz Biotechnology, Santa Cruz, CA) and insulin (Dako). p57Kip2 staining was detected with the streptavidin biotin-peroxidase kit (Zymed Laboratories, South San Francisco, CA) and aminoethylcarbazole as substrate. Insulin was stained using the streptavidin biotin-alkaline phosphatase kit (Zymed Laboratories) using the substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. To prevent cross-reactivity, avidin-biotin blocking kit was used before incubation with anti-insulin antibody. As negative control, slides underwent the same procedure but were incubated with PBS without anti-p57Kip2 antibody.
Genetics (screening for mutations in ABCC1 and KCNJ11)
All exons and flanking introns of the SUR1 gene and the entire Kir6.2 open reading frame was subjected to PCR amplification and tested for small deletions, insertions, or point mutations using denaturing HPLC (Wave 3500; Transgenomic, Omaha, NE). Samples showing deviating chromatografic patterns were sequenced using the DYEnamic* ET dye terminator kit (Amersham Biosciences, Arlington Heights, IL) and analyzed on an automated MegaBaceTM DNA sequencer (Amersham Biosciences).
Functional studies: tissue preparation
After surgery, islets of Langerhans were isolated using a controlled collagenase digestion procedure and were dispersed into single cells as described previously (22, 23). Dispersed cells were incubated at 37 C in a humidified atmosphere of 5% CO2/air mixture for up to 4 d and were maintained under standard tissue culture conditions in RPMI 1640 medium (Sigma) supplemented with 10% v/v fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Electrophysiology
All data were obtained using cell-attached or inside-out recording configurations of the patch-clamp technique as described previously (24). The pipette contained a standard NaCl-rich bathing solution containing the following (in mM): 140 NaCl, 4.7 KCl, 2.5 CaCl2, 1.13 MgCl2, 10 HEPES, and 2.5 glucose (pH 7.4 with NaOH). The bath solution contained the following (in mM): 140 KCl, 10 NaCl, 1.13 MgCl2, 1 EGTA, 2.5 glucose, and 10 HEPES (pH 7.2 with KOH) for all recordings.
Immunohistocytochemistry
Isolated cells were cultured with poly-D-lysine-coated coverslips (100 µg/ml) for 24 h in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2% penicillin/streptomycin. The cells were fixed by applying Zamboni’s fixative for 50 min and blocked with 1% goat serum in PBS with 0.1% Triton X-100 for 30 min. They were incubated overnight at 4 C with a polyclonal anti-SUR1 antibody used at 1:300 dilution. The cells were rinsed, and the secondary antibody, goat antirabbit FITC-conjugated IgG, was applied at 1:150 dilution (Sigma).
Ca2+ signaling
Changes in the cytosolic Ca2+ concentration were monitored by digital imaging microfluorimetry (Roper Scientific, Marlow, Bucks, UK) of cells loaded with fura 2-AM to a final concentration of 20 µM for 30–40 min at 37 C at which the coverslip formed the base of a perifusion chamber (Warner Instruments, Edenbridge, Kent, UK).
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Results |
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The results of microsatellite marker analysis for markers D11S1984, TH, and D11S1318 are shown in Figure 2. Dosage analysis of patient showed evidence in favor of the paternally derived allele at a ratio of 1.7:1 and 1.5:1 for markers D11S1984 and TH, respectively. The ratios obtained were consistent with a diagnosis of mosaic paternal isodisomy (Figs. 1
and 2).
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Histological examination of the resected pancreas showed throughout the specimen a marked proliferation of endocrine tissue forming irregular nodules rather than discrete islets. These nodules of endocrine tissue contained somatostatin- and glucagon-producing cells in the periphery. In addition, throughout the islets, there were pancreatic polypeptide-immunoreactive cells. Immunostaining for proinsulin was strong in these nodules, but insulin immunostaining, although present, was weak. An antibody against low-molecular-weight cytokeratin showed the presence of the remaining ascinar tissue but also demonstrated that ductular structures were not a prominent feature in the proliferating nodules of endocrine tissue. Figure 3 shows the histological appearance of the resected pancreas.
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p57Kip2 stain was clearly positive in ß-cells with very few positively stained nuclei outside the islets. p57Kip2 staining is shown in Figure 4.
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Denaturing HPLC analysis of ABCC8 and KCNJ11 genes revealed deviating banding patterns in SUR1 exons 16, 23, and 33; additionally, three variants were disclosed in Kir6.2. Sequencing revealed all patterns to be polymorphisms seen in the general population at large. Both of the polymorphisms in the six codons of the ABCC8 and KCNJ11 genes were seemingly paternally derived, and no maternal polymorphisms were seen in the ABBC8 and KCNJ11 genes. No silent mutations or rare intron variations were found.
Functional studies
Figure 5 summarizes the expression of KATP channels in isolated cell membrane patches by electrophysiology. In control human ß-cells, KATP channels are present, and the average current value per patch of membrane was 25.5 ± 1.48 pA (n = 263). In contrast, no KATP channels were recorded in the patient tissue. Similar data were obtained in the HI ß-cells with truncations of the C-terminal domain of SUR1 (Fig. 5A), whereas in other HI patient tissue, a modest level of channel activity was recorded. The loss of functional KATP channels in BWS ß-cells was correlated with immunofluorescence data using a SUR1 antibody that showed a perinuclear pattern of staining (Fig. 5B). We also examined the control of Ca2+ signaling events in BWS ß-cells (Fig. 6). These data showed that responses to glucose and tolbutamide were impaired. For example, 10 mM glucose induced a 34 ± 4 nM rise in cytosolic Ca2+ in 26 of 38 experiments, whereas only 31 of 66 cell clusters responded to 0.1 mM tolbutamide ([Ca2+]i = 27 ± 3 nM). Most cells, however, were responsive to 40 mM KCl-induced depolarization of the cell membrane, suggesting that voltage-gated Ca2+ channels were unaffected by the loss of KATP channels.
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Discussion |
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We were also able to show impaired responses of BWS ß-cells to glucose and tolbutamide, which is consistent with the ion channel data. However, the fact that some cells were able to respond to tolbutamide and glucose does imply an ability of cells to express normal channels. Collectively, we would suggest that hyperinsulinism is related to aberrant expression of sufficient numbers of channels at the cell surface. HI-causing mutations in the ABCC8 and KCNJ11 genes (encoding SUR1 and Kir6.2, respectively) impair the function of the KATP channel by affecting channel density, channel expression, channel trafficking from the Golgi apparatus and endoplasmic reticulum, channel gating properties, and channel activity in response to changes in the concentrations of intracellular nucleotides (26, 27). Mutations in these genes are, however, only found in about 50% of patients with HI (28). Despite extensive search, no mutations were found in the genes ABCC8 or KCNJ11 in this patient. Mutations in the promoter region of ABCC8, or larger, partial deletions of ABCC8 or KCNJ11, were, however, not excluded with the methods in use. Another possible genetic cause could be a mutation in an unknown gene affecting trafficking of the SUR1-Kir6.2 complex.
Histological examination of the pancreas showed strong proinsulin and weak insulin immunostaining, suggesting that the ß-cells were secreting large amounts of insulin. The proliferation of endocrine tissue was reminiscent of the appearances seen in Fo-HI. However, in this patient, proliferating islets were seen throughout the pancreas, and ductular structures were not a feature within these nodules and there was no evidence of fibrosis. Thus, the changes seen in our patient are different from those seen in typical Fo-HI. Because p57Kip2 is paternally imprinted in human pancreatic ß-cells and there is the loss of expression in Fo-HI, the fact that p57Kip2 protein expression was readily demonstrated throughout the pancreas also excludes focal forms of the disease.
The genetics of the BWS in this patient showed that, in the lymphocytes, the paternally derived allele had a ratio of 1.7:1, 1.5:1, and 1.2:1 for markers D11S1984, TH, and D11S1318, respectively. The ratios obtained were regarded as evidence of mosaic paternal isodisomy. The expression of p57Kip2 in the ß-cells suggests that, at least in the ß-cells, there was no loss of the maternal 11p15.5 region, thus further supporting the evidence of mosaic paternal UPD. The inheritance patterns of the polymorphisms in the ABCC8 and KCNJ11 genes gave, however, evidence of paternal heterodisomy in the region 11p15.1 and loss of maternal 11p15.1. Accordingly, our patient may represent a unique case of mosaic paternal uniparental isodisomy in the 11p15.5 region and paternal uniparental heterodisomy of the 11p15.1 region.
The mechanism(s) in which paternal uniparental heterodisomy of the 11p15.1 region causes a KATP trafficking defect remains to be elucidated. Both of the ABCC8 and the KCNJ11 alleles were paternally derived, and the father was healthy. In one of the alleles of the child, the areas 11p15.5 and 11p15.1 arose from two different paternal alleles, suggesting a break of continuity with a possible undiscovered partial gene deletion in ABCC8 in the area of exon 33 to exon 39, in which no polymorphisms were seen. This could have a dominant action and explain the persistent, severe hyperinsulinemic phenotype and the impaired trafficking of the SUR1-Kir6.2 complex.
Another possibility is a mosaicism of paternal isodisomy and paternal heterodisomy in 11p15.1, which would allow a recessive undiscovered mutation in ABCC8 or KCNJ11 to become homozygous in a majority of the cells. A search for mutations in the ABCC8 promoter region will be performed to investigate this possibility.
It is suggested that one of these genetic errors in the 11p15.1 region gave rise to a KATP trafficking defect and persistent, severe HI, together with the typical mosaic paternal uniparental isodisomy in the 11p15.5 region, resulting in a Beckwith-Wiedemann phenotype and an atypical diffuse islet cell histology.
In summary, we have described a novel case of BWS with mosaic paternal UPD for 11p15 in which persistent hyperinsulinism was found to be caused by abnormalities in KATP channels of the pancreatic ß-cell. No mutations were found in the genes ABCC8 and KCNJ11 encoding for the two subunits SUR1 and Kir6.2, respectively, of the KATP channel.
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Footnotes |
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First Published Online April 5, 2005
Abbreviations: BWS, Beckwith-Wiedemann syndrome; Fo-HI, focal form of hyperinsulinism in infancy; HI, hyperinsulinism in infancy; KATP channel, ATP-sensitive K+ channel; TH, tyrosine hydroxylase; UPD, uniparental disomy.
Received January 25, 2005.
Accepted March 24, 2005.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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  R R Kapoor, S E Flanagan, C James, J Shield, S Ellard, and K Hussain Hyperinsulinaemic hypoglycaemia Arch. Dis. Child., June 1, 2009; 94(6): 450 - 457. [Abstract] [Full Text] [PDF]
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K. Hussain, O. Blankenstein, P. De Lonlay, and H. T Christesen Hyperinsulinaemic hypoglycaemia: biochemical basis and the importance of maintaining normoglycaemia during management Arch. Dis. Child., July 1, 2007; 92(7): 568 - 570. [Full Text] [PDF]
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